2′-O-methoxy-ethyl (MOE), Affinity Plus Locked Nucleic Acid, 2′-O-methyl RNA, and 5-methyl dC
State-of-the-art antisense design employs chimeras with both DNA and modified RNA bases. The use of modified RNA, such as 2′-O-methoxy-ethyl (2′-MOE) RNA, 2′-O-methyl (2′OMe) RNA, or Affinity Plus Locked Nucleic Acid bases in chimeric antisense designs, increases both nuclease stability and affinity (Tm) of the antisense oligo to the target mRNA [3–5]. However, these modifications do not activate RNase H cleavage. The preferred antisense strategy is a "gapmer" design which incorporates 2′-O-modified RNA or Affinity Plus Locked Nucleic Acid bases in chimeric antisense oligos that retain an RNase H activating domain. As unmodified DNA is susceptible to rapid degradation by endo- and exo-nucleases and many 2′-O-modified RNA (such as 2′OMe RNAs and Affinity Plus Locked Nucleic Acid bases) are sensitive to exonuclease degradation, we recommend phosphorothioate modification of the antisense oligo to provide stability. Phosphorothioate linkages also promote binding to serum proteins which increases the bioavailability of the antisense oligo and facilitates productive cellular uptake.
It can also be beneficial to substitute 5-methyl-dC for dC in the context of CpG motifs. Substitution of 5-methyl dC for dC will slightly increase the Tm of the antisense oligo. Use of 5-methyl dC in CpG motifs can also reduce the chance of adverse immune response to Toll-like receptor 9 (TLR9) in vivo. We recommend standard desalt purification for most antisense applications. For use in live animals, higher purity oligos may be required. In these instances, HPLC purification combined with Na+ salt exchange followed by end-user ethanol precipitation of the antisense oligo is recommended to mitigate toxicity from residual chemicals that may carry over during synthesis.